PROJECT SUMMARY/ABSTRACT Tumor-associated macrophages (TAMs) are often the dominant immune cell type found in solid human tumors, and while their presence is correlated with poor disease prognosis, it remains to be determined what triggers and controls their differentiation. Using a murine model of spontaneous breast cancer that closely recapitulates human disease, we have recently demonstrated that TAMs are derived from monocytes, their differentiation relies on Notch signaling, and in their absence, tumor growth is slowed and T cells in the tumor express more effector molecules and fewer exhaustion markers, suggesting an immunosuppressive role for TAMs. My proposed work aims to further explore the differentiation program and function of TAMs in the tumor microenvironment. My preliminary findings demonstrate that TAMs specifically express high levels of the transcription factor interferon regulatory factor 8 (IRF8), and in the absence of IRF8, TAM differentiation is blocked. IRF8 is known to be a key lineage-determining factor for other myeloid cell populations and can mediate expression of genes related to antigen-presentation, thus influencing T cell responses. In this proposal, I aim to characterize how IRF8 mediates TAM differentiation and function using mouse genetics and mouse models of disease. I will determine what external cues drive IRF8 expression and what genetic programs IRF8 controls in TAMs using genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) and RNA-sequencing. Through loss-of-function studies and ex vivo cell culture assays, my work will directly measure the potential IRF8-mediated TAM immunosuppressive activities on T cells and other immune cells in the tumor microenvironment. The outlined proposal, using definitive approaches, will generate conclusive, meaningful data and will broaden our understanding of TAM function and identity. My findings may uncover novel avenues for cancer immunotherapies to manipulate TAMs, boosting endogenous anti-tumor responses and providing critical new tools in the fight against cancer.